Hardip Sandhu

Molecular basis of cancer-therapy-induced cardiotoxicity: introducing microRNA biomarkers for early assessment of subclinical myocardial injury.

Development of reliable biomarkers for early clinical assessment of drug-induced cardiotoxicity could allow the detection of subclinical cardiac injury risk in vulnerable patients before irreversible damage occurs. Currently, it is difficult to predict who will develop drug-induced cardiotoxicity owing to lack of sensitivity and/or specificity of currently used diagnostics. miRNAs are mRNA regulators and they are currently being extensively profiled for use as biomarkers due to their specific tissue and disease expression signature profiles. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable tool to allow prognosis of patients at risk of cardiovascular injury, alteration of a treatment regime or the introduction of an adjunct therapy in order to increase the long-term survival rate of patients treated with cardiotoxic drugs.

Comparison of MEK/ERK pathway inhibitors on the upregulation of vascular G-protein coupled receptors in rat cerebral arteries

Organ culture is an in vitro method for investigating cellular mechanisms involved in upregulation of vasocontractile G-protein coupled receptors. We hypothesize that mitogen-activated-protein kinase (MEK) and/or extracellular-signal-regulated kinase (ERK) specific inhibitors will attenuate the G-protein coupled receptor expression following organ culture. Rat cerebral arteries were incubated 48h in the presence of MEK/ERK specific inhibitors U0126, PD98059, SL327, or AG126 for different time periods. Contractile responses by activation of endothelin receptor type A and type B, serotonin receptor 5-HT(1B), prostanoid TP receptor, and angiotensin II receptor type 1 and type 2 were investigated. Results were verified by measurement of mRNA with real time PCR and by protein immunohistochemistry. Organ culture induced transcriptional upregulation of endothelin ET(B) receptor and of serotonin 5-HT(1B) receptor on translational level and increased respective contractions. The prostanoid TP receptor mediated contraction curve was left-wards shifted by organ culture. Organ culture was associated with elevated pERK1/2 in the vascular smooth muscle cells: the MEK1/2 inhibitor U0126 attenuated the endothelin ET(B) receptor mediated contraction at post-translational level or by changing the receptor affinities. The serotonin 5-HT(1B) receptor and prostanoid TP receptor mediated contractions were abolished by U0126. Administration of U0126 6h after start of incubation blocked the receptor upregulation. In conclusion, MEK specific inhibitor U0126 is a potent inhibitor of G-protein coupled receptor alteration seen during organ culture. Given the ability to inhibit G-protein coupled receptor alteration at the clinically relevant time-point 6h post incubation makes it an attractive therapeutic agent for in vivo studies.

Upregulation of contractile endothelin type B receptors by lipid-soluble cigarette smoking particles in rat cerebral arteries via activation of MAPK

Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ET(B)) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) mediate the upregulation of contractile endothelin receptors in the cerebral arteries. Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSP with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-κB specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ET(B) receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ET(B) receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively. Results show that organ culture per se induced transcriptional upregulation of contractile ET(B) receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ET(B) receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ET(B) receptors. Thus, the MAPK-mediated upregulation of contractile ET(B) receptors in cerebral arteries might be a pharmacological target for the treatment of smoke-associated cerebral vascular disease like stroke.

Alteration in contractile G-protein coupled receptor expression by moist snus and nicotine in rat cerebral arteries

The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET(B), serotonin 5-HT(1B), and thromboxane A(2) TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET(B), 5-HT(1B), and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25ng nicotine/ml) lowered the 5-HT(1B) receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250ng nicotine/ml) increased the ET(B) receptor-mediated contraction. The combined 5-HT(1B) and 5-HT(2A) receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or nicotine (250ng nicotine/ml). Only the DSS group showed that the increase of 5-HT(1B) receptor-mediated contraction occurred at the transcriptional level, demonstrated by an increased mRNA expression for the receptor. Thus, snus and nicotine alter the GPCR expression in the cerebral arteries, which may be involved in cerebral vascular disease.

Attenuation of Sunitinib-induced cardiotoxicity through the A3 adenosine receptor activation

ABSTRACT Sunitinib is an anti‐cancer tyrosine kinase inhibitor associated with severe cardiotoxic adverse effects. Using rat Langendorff heart model and human acute myeloid leukaemia 60 (HL60) cell line we detected the involvement of protein kinase C (PKC) &agr; during Sunitinib‐induced cardiotoxicity and the effect of Sunitinib on cancer progression. The cardioprotective and anti‐cancer properties of the A3 adenosine receptor agonist 2‐chloro‐N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) were investigated. The cardiac effect of Sunitinib (1 &mgr;M) and IB‐MECA (1 nM) treatment was measured through haemodynamic and infarct size assessment. The cytotoxic effect of Sunitinib (0.1 – 10 &mgr;M) and IB‐MECA (10 nM – 10 &mgr;M) on HL60 cells was assessed using the methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay technique. Myocardial injury associated microRNAs (miR‐1, miR‐27a, miR‐133a and miR‐133b) and cancer associated microRNAs (miR‐15a, miR‐16‐1 and miR‐155) were profiled by qRT‐PCR in the cardiac tissue and HL60 cells, while phosphorylated PKC&agr; levels were measured by Western Blot analysis. Sunitinib treatment increased infarct size and decreased left ventricular developed pressure and heart rate. Co‐treatment of IB‐MECA reversed the myocardial injury produced by Sunitinib administration. IB‐MECA did not jeopardize the anti‐cancer effect of Sunitinib in HL60 cells. The expression signature of the specific microRNAs in cardiac tissue and HL60 cells showed an altered expression profile when treated with Sunitinib and IB‐MECA. pPKC&agr; levels were increased by Sunitinib treatment in cardiac tissue and HL60 cells and co‐administration of IB‐MECA attenuated this increase in the cardiac tissue. This study reveals that A3 adenosine receptor activation by IB‐MECA attenuates Sunitinib‐induced cardiotoxicity through the involvement of PKC&agr;.

Involvement of mitogen activated kinase kinase 7 intracellular signalling pathway in Sunitinib-induced cardiotoxicity

The tyrosine kinase inhibitor Sunitinib is used to treat cancer and is linked to severe adverse cardiovascular events. Mitogen activated kinase kinase 7 (MKK7) is involved in the development of cardiac injury and is a component of the c-Jun N-terminal kinase (JNK) signal transduction pathway. Apoptosis signal-regulating kinase 1 (ASK1) is the upstream activator of MKK7 and is specifically inhibited by 2,7-dihydro-2,7-dioxo-3H-naphtho[1,2,3-de]quinoline-1-carboxylic acid ethyl ester (NQDI-1). This study investigates the role of ASK1, MKK7 and JNK during Sunitinib-induced cardiotoxicity. Infarct size were measured in isolated male Sprague-Dawley rat Langendorff perfused hearts treated for 125 min with Sunitinib in the presence and absence of NQDI-1. Left ventricular cardiac tissue samples were analysed by qRT-PCR for MKK7 mRNA expression and cardiotoxicity associated microRNAs (miR-1, miR-27a, miR-133a and miR-133b) or Western blot analysis to measure ASK1/MKK7/JNK phosphorylation. Administration of Sunitinib (1 μM) during Langendorff perfusion resulted in increased infarct size, increased miR-133a expression, and decreased phosphorylation of the ASK1/MKK7/JNK pathway compared to control. Co-administration of NQDI-1 (2.5 μM) attenuated the increased Sunitinib-induced infarct size, reversed miR-133a expression and restored phosphorylated levels of ASK1/MKK7/JNK. These findings suggest that the ASK1/MKK7/JNK intracellular signalling pathway is important in Sunitinib-induced cardiotoxicity. The anti-cancer properties of Sunitinib were also assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Sunitinib significantly decreased the cell viability of human acute myeloid leukemia 60 cell line (HL60). The combination of Sunitinib (1 nM-10 μM) with NQDI-1 (2.5 μM) enhanced the cancer-fighting properties of Sunitinib. Investigations into the ASK1/MKK7/JNK transduction pathway could lead to development of cardioprotective adjunct therapy, which could prevent Sunitinib-induced cardiac injury.

Cardiovascular Diseases Associated with Pregnancy: Early Assessment Using Non-Invasive MicroRNA Profiling

In some cases pregnancies are associated with severe cases of cardiovascular diseases (CVDs). The early detection and proper treatment of CVDs during maternity is detrimental to the health outcome and wellbeing of both mother and child. Unfortunately, both the detection rate and assessment of CVDs during pregnancies are unsatisfactory. Currently, the messenger RNA (mRNA) regulators called microRNAs (miRNAs) are being extensively profiled for use as clinical CVDs biomarkers due to their specific tissue and disease expression signature profiles. The identification and development of reliable biomarkers for early clinical assessment of CVDs during pregnancy could allow the detection of sub-clinical cardiac injury risk in vulnerable pregnant patients before irreversible damage occurs. CVDs specific miRNA biomarkers could provide the clinicians with a valuable tool to allow prognosis of patients at risk of cardiovascular injury and the introduction of therapy and intervention in order to increase health outcome and survival rate of both the mother and child.

20 Cardioprotection During Chemotherapy: A Case Study to Understand Intracellular Mechanisms to Combat the Cardiotoxicity of Sunitinib

Tyrosine Kinase inhibitor Sunitinib mediates its apoptotic anti-cancer effects by inhibiting intracellular signalling involved in tumour angiogenesis and cell proliferation. Unfortunately Sunitinib has revealed unwanted cardiotoxicity side-effects and investigations into cardioprotective adjunct therapy agents are critical. A3 adenosine receptor agonists (A3AR) have been shown to have powerful cardioprotective effects against myocardial injury (MI). We studied the effect of A3AR agonist IB-MECA in preventing MI following Sunitinib treatment and assessed whether IB-MECA jeopardized the anti-cancer/apoptotic effect of Sunitinib in cancer cells. The associated intracellular signalling pathway through Protein Kinase C α/β (PKC α/β) and microRNA expression signatures were investigated. HL60 cells were incubated with increasing concentrations of Sunitinib (0.1-10 μM) ± IB-MECA (1 nM) for 24h and cell viability (CV) was assessed. Langendorff hearts underwent Sunitinib (0.3 μM) ± IB-MECA (1 nM) treatment and MI assessment. Phosphorylated expression levels of Protein Kinase C α/β were assessed by Western Blot analysis and rtPCR analysis revealed the microRNA expression signature. Sunitinib decreased HL60 cell viability and increased MI compared to vehicle (Vehicle: CV = 102.0 ± 1.4%; MI = 8.2 ± 3.6%; Sunitinib: CV(10 μM) = 43.2 ± 6.3%, p = 0.02; MI(0.3 μM) = 30.1 ± 1.5%, p < 0.001). Addition of IB-MECA did not alter the apoptotic effect of Sunitinib, however, IB-MECA attenuated the Sunitinib-induced MI (Sunitinib + IB-MECA: CV(10 μM) = 37.8 ± 6.4%; MI(0.3 μM) = 20.4 ± 2.8%, p < 0.01). The IC50-values of Sunitinib ± IB-MECA did not change significantly (Sunitinib IC50-value = 8.4 ± 1.3; Sunitinib + IB-MECA IC50-value = 7.0 ± 1.6) (n = 5–6). This study reveals for the first time that A3AR activation improves myocardial survival by attenuating Sunitinib induced MI without interfering with the anti-tumour efficacy of Sunitinib. A3AR associated signalling pathways could be important in the development of adjunctive chemotherapy treatment.

Ageing alters the severity of Sunitinib-induced cardiotoxicity: Investigating the mitogen activated kinase kinase 7 pathway association

Anti-cancer drug Sunitinib is linked to adverse cardiovascular events, which have shown to involve mitogen activated kinase kinase 7 (MKK7) pathway. Sunitinib-induced cardiotoxicity in 3, 12 and 24 months old male Sprague-Dawley rats and MKK7 expression and activation was investigated using the Langendorff perfused heart model followed by Western blot analysis. Cardiac function and infarct size were measured during/after 125 min of Sunitinib treatment. Left ventricular cardiac samples were analysed by qRT-PCR for expression of MKK7 mRNA and cardiac injury associated microRNAs. Infarct size was increased in all Sunitinib treated age groups. Haemodynamic alterations were observed following Sunitinib administration. Left ventricular developed pressure (LVDP) was decreased in all age groups, while heart rate (HR) was decreased in 3 and 12 months groups. Sunitinib treatment decreased the expression of miR-27a in all age groups, while miR-133a and miR-133b levels were increased in 3 months and decreased in 24 months groups. MKK7 mRNA and p-MKK7 levels were decreased in the 3 months group after Sunitinib treatment. MKK7 mRNA level was increased in 24 months group and p-MKK7 levels were increased in 12 months group following Sunitinib treatment. This study highlights the importance and impact of ageing and anti-cancer therapy-induced cardiotoxicity.

198 Involvement of Mitogen Activated Kinase Kinase 7 in Tyrosine Kinase Inhibitor Induced Myocardial Injury

Introduction Mitogen Activated Protein (MAP) kinase signalling cascades play significant roles in the development of cardiac hypertrophy in response to external stresses. Mitogen activated kinase kinase 7 (MKK7) is a selective upstream regulator of the c-Jun N-terminal kinase (JNK) pathway. Specific over expression of MKK7 in hearts has been shown to increase the sensitivity of cardiomyocytes to external stresses. We investigated the expression and activation levels of MKK7 at mRNA and protein level; in particular, investigating the level of MKK7 phosphorylation in response to external stresses induced by the tyrosine kinase receptor inhibitor Sunitinib. Although this Sunitinib has been shown to be highly effective in the treatment of cancers such as metastatic renal-cell carcinoma and gastrointestinal stromal tumours, adverse cardiovascular events have been reported in treated patients. Methods Langendorff perfused hearts were perfused with vehicle (normoxic control) or Sunitinib for 120 min and either stained with triphenyl-tetrazolium chloride to assess the level of infarct to risk size (n = 4) or analysed by real time PCR for the expression profile of MKK7 mRNA (n = 6) or analysed by western blot to assess the level of MKK7 activation (n = 4). Results TTC staining revealed that Sunitinib causes a significant increase in infarct size compared to a normoxic control group (vehicle=5.4 ± 2%; Sunitinib=46 ± 4.5%, P < 0.0001). mRNA levels of MKK7 remained unchanged. However, an increase in phosphorylated MKK7 (p-MKK7) levels were detected. Conclusion The increase in p-MKK7 expression directly correlates with Sunitinib induced cardiotoxicity, suggesting that MKK7 could be an important intracellular signalling protein involved in tyrosine kinase inhibitor induced cardiotoxicity.